A big problem currently exists in studying the dynamics of complex biological systems and processes occurring in them, where there is spatial heterogeneity. Such processes include, in particular, blood coagulation, complement, apoptosis, digestion, fibrinolysis, in which proteolytic enzymes (proteinases) play a key role.
Concentration of proteolytic enzymes can be measured if this value is unchanging in time and is the same at all points of the sample being analyzed with the use of a specific fluorogenic substrate or a chromogenic substrate. Currently, methods measuring changes in concentration over time are used in fundamental research and in diagnostics of functional failures of the corresponding biological systems. To determine blood coagulation disorders, a test of thrombin generation in plasma is now used, which was disclosed in the basic paper by Hemker H C, Wielders S., Kessels H., Beguin S., Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential, J Thromb Haemost. 1993, Oct. 18, 70 (4):617-24. The test has demonstrated many advantages over traditional coagulation tests, but it is spatially uniform, i.e. a homogeneous environment is studied. This doesn't correspond to the situation in the organism described below.
FIG. 1 schematically represents the spatial concept of regulation of blood coagulation. Coagulation is activated by cells expressing a transmembrane protein—tissue factor, a nonenzymatic co-factor being a coagulation factor (left), —and propagates deep into the plasma. The generation of thrombin is regulated by activated factor X (factor Xa, serine proteinase), a limiting component of prothrombinase. The coagulation near the activator (the initiation phase) is determined solely by production of factor Xa by extrinsic tenase—a complex of tissue factor and serine proteinase of factor VIIa. However, factor Xa is inhibited rapidly and cannot diffuse far away from the activator. Therefore, during the clot propagation phase it is formed by intrinsic tenase. The limiting component of the intrinsic tenase, clotting factor IXa, is produced by extrinsic tenase. In contrast to factor Xa it is inhibited slowly and therefore diffuses far away. Following further increase of the clot, additional factor IXa is produced by factor XIa, which in turn is produced by thrombin within a positive feedback loop. The clot formation stops due to the action of thrombomodulin: a negative feedback loop activates protein C, which stops propagation of thrombin by destruction of factors Va and VIIIa (see Panteleev M. A., Ovanesov M. V., Kireev D. A., Shibeko A. M, Sinauridze E. I., Ananyeva N. M., Butylin A. A., Saenko E. L., and Ataullakhanov F. I., Spatial Propagation and Localization of Blood Coagulation Are Regulated by Intrinsic and Protein C Pathways, Respectively, Biophys J. 2006 Mar. 1; 90(5):1489-500). Despite the fact that some details of this concept can be corrected, the key role of diffusion processes and spatial heterogeneity in clotting is undisputed (Hoffman M., Monroe D M 3rd, A cell based model of hemostasis, Thromb Haemost. 2001 June; 85(6):958-65).
Thrombin is the key enzyme of the blood coagulation system. It catalyzes the main reaction—the conversion of fibrinogen into fibrin. In addition, thrombin activates coagulation factors V, VIII, VII, XI, XIII, protein C, platelets, thrombin-activated fibrinolysis inhibitor. In coagulation, the volume of thrombin produced is 10-100 times bigger than the one of the other proteinases, which facilitates its detection.
In the thrombin-catalyzed reaction, fibrinogen is converted into fibrin, which polymerizes and thus jellifies blood plasma.
Studies of blood coagulation are of great practical interest because they do not only allow certain diseases to be diagnosed, but also make it possible to assess the activity of drugs affecting blood coagulation parameters.
Appearance of chromogenic and then fluorogenic substrates accelerated the coagulation studies. Such synthetic substrate is a molecule that is recognized and cut by proteolytic enzyme. Cutting leads to cleavage from the substrate of a signaling molecule also referred to as “mark”. The mark either changes the optical density of the solution (chromogenic or coloring substrate), or can fluoresce when illuminated (fluorogenic substrate), or can spontaneously emit light without external excitation (chemiluminescent mark). Substrates for thrombin can be added directly into plasma, and the signal (optical density or light intensity) appearing in coagulation can be recorded. The rate of increase of the signal is proportional to concentration of thrombin. Thrombin dependence on time is obtained from the experimental relation between signal and time by simple differentiation and calculation of thrombin concentration based on the substrate cleavage rate using the calibration curve obtained by adding to a buffer or plasma under analysis of known concentrations of thrombin or another calibrator (e.g. complex of thrombin and alpha2-macroglobulin).
Various methods and devices are known from the background art for determining blood coagulation parameters in-vitro. However, all known methods and devices are usually designed to work within homogeneous systems, where a sample of blood or plasma is uniformly mixed with an activator, which substantially distinguishes these systems from the system in-vivo being a complex heterogeneous environment.
In a well-known model, the in-vitro systems' conditions of the coagulation process are fundamentally different from the conditions in which the clot is formed in a living organism. It is known that in the circulatory system of humans and animals a clot is formed not in the entire volume of blood plasma, but strictly locally, i.e. in a small area near the damaged blood vessel wall. Clotting in the body is not uniform. Formation of a clot occurs in space. It is induced by extrinsic tenase on a damaged vessel wall, propagates with participation of prothrombinase on the activated platelets in the bulk of plasma, and is inhibited by reactions involving thrombomodulin on healthy endothelium. In this case coagulation factors are naturally distributed in a small volume of plasma, and a clot forms therein. This reflects the basic defense mechanism of the hemostatic system: maintenance of integrity of the bloodstream through formation of a blood clot at the site of injury. These processes cannot be adequately studied using the methods carried out in a homogeneous medium.
Thus, there is currently a problem of experimental modeling of blood coagulation in-vitro, as it is desirable to more fully simulate the spatial situation in which a blood clot coagulates directly in a blood vessel. The problem exists both for fundamental studies of thrombosis and hemostasis, and for application-specific diagnostic and pharmacological tasks.
The problem of determining the changes of the proteolytic enzyme concentration in time and space, i.e. at different points within the volume of the test sample, has not been solved yet.
Recently, devices have been used which allow taking into account the spatial heterogeneity and diffusion of coagulation factors. In such devices, coagulation occurs in a cuvette containing recalcified plasma. The activator is a surface with immobilized clotting activator, e.g. tissue factor. Coagulation begins upon contact of the activator with the plasma and then propagates deeper into the plasma; it can be observed by light scattering from the growing clot.
From the background art, we know a device for investigation of coagulation characteristics of blood and its components (patent RU 2395812, cl. G01N33/49, published on 27, Jul. 2010) comprising a thermostatically controlled chamber which accommodates a cuvette with a test plasma sample and a coagulation activator, such as thromboplastin (coagulation tissue factor) applied on the insert put into the cuvette, LEDs for lightening the cuvette content and a clot forming near the activator, a digital camera recording the growing clot, and a computer for processing the obtained data.
This device allows implementing a method which involves recording only the process of formation of a fibrin clot being the final product of the coagulation system.
We also know a method and an apparatus for monitoring the spatial fibrin clot formation (International application PCT/CH2007/000543, cl. G01N33/49, published on 7, May 2009, publication number WO 2009/055940).
The apparatus comprises a cuvette used for photometric analysis, comprising a chamber, an insert and an activator, a thermostat wherein the said cuvette is placed. The coagulation activator is located on the bottom edge of the insert. The coagulation activator is a physiological activator, such as tissue factor, or a non-physiological activator, such as glass. The cuvette is made of light-transmitting polystyrene.
The device allows in-vitro monitoring of formation and/or lysis of a fibrin clot and comprises the following steps:
placing in a cuvette one or more plasma samples depending on the number of wells, and
inserting into the cuvette an insert with an activator and bringing plasma into contact with a coagulation activator (in case of clot formation), and
recording the growth of the fibrin clot as a function of time and distance, or
placing in the cuvette one or more plasma samples containing one or more fibrin clots,
bringing plasma into contact with a fibrinolysis activator (in case of clot lysis), and
recording the lysis of the fibrin clot as a function of time and distance.
The principal advantage of the method and apparatus for monitoring spatial fibrin clot formation is that only a small volume of plasma is needed. With as small amount as 20 μl (instead of 300 up to 1500 μl, i.e. 75-fold less than in the other similar system reported previously, and 5-fold less than the minimal plasma amount required for standard clotting assays), reliable high resolution results can be produced. This apparatus allows implementing a method which involves recording only the formation process of a fibrin clot being the final product of the coagulation system.
The disadvantage of the abovementioned device and method is the formation of gas bubbles in the cuvette within the registration area when the test samples are heated, which distorts the light scattering signal from the fibrin clot.
Light sources having only one wavelength, such as red light, prevent the study of spatiotemporal distribution of fluorescent substances.
Moreover, the device and method described above do not provide the possibility to record the process of formation and spatial distribution of separate clotting factors, such as IIa, Xa, VIIa, XIa, which regulate the process of spatial fibrin clot growth.
Most closely related to the present method and device are the apparatus and method disclosed in article by Kondratovich A. Y., Pohilko A. V. and Ataullahanov F. I., Spatiotemporal Dynamics of Contact Activation Factors of Blood Coagulation, Biochim Biophys Acta. 2002 Jan. 15; 1569 (1-3):86-104).
To carry out the abovementioned method, platelet-poor plasma is used. Distributions of factor XIa and kallikrein of the studied plasma sample are determined by recording the indigo emission of 7-amino-4-methyl-coumarin (AMC), product of cleavage of fluorogenic substrates specific to these factors.
Prior to the measurements, a substrate was added to each test plasma sample, and the sample was stirred at 37 degrees C.; pH of the medium was maintained at 7.4 at this temperature.
FIG. 2 shows schematically a device used to implement the method. The device comprises a polystyrene dish 1 containing the studied blood plasma sample 2. The substrate is added to plasma 2. The tip 5 of the glass capillary was used as a coagulation activator. The device also contains: light source 6—a mercury lamp, thermostat 7, glass filter 8, semitransparent mirror 9, digital camera 10, fluorescing plastic label 12; the device is plugged to computer 11.
Activation of clotting factors was studied in a two-dimensional (flat) medium, that is, in a thin layer of unstirred plasma. Dish 1 was transferred to thermostat 7 at 37 degrees C., and the activator was quickly lowered so that capillary end 5 would be submerged into the plasma.
The coagulation factor activated by contact with glass cleaved the substrate, giving rise to formation of AMC. Fluorescence of AMC was recorded as follows. The plasma sample was lightened by light from light source 6 reflected from semitransparent mirror 9. Filters 8 blocked the visible portion of the light source spectrum. AMC fluorescence was recorded by digital camera 10 mounted behind the semitransparent mirror. The recorded field of view measured 9.0×6.5 mm. Blue channel of the RGB output signal of the camera spanned the entire range of AMC fluorescence. The image data was continuously transferred to computer 11, displayed on its monitor and saved at specified intervals. Piece of fluorescing plastic 12 was fixed beneath thermostat 7 so that its image was always in the field of view of the camera; this was used for calibration and for taking into account the light variation.
In analysis of the images, a radial line beginning in the activator's center was selected. The spatiotemporal distribution of AMC concentration along the line was determined by specific software (FIG. 3), and the spatiotemporal distribution of clotting factor concentration was recovered on its basis.
Disadvantages of this method include the ability of measuring only contact activation factors (particularly, factors XIa, XIIa, kallikrein) with poor ability of differentiating between contributions of these factors into signal and without the possibility of measuring the spatial dynamics of coagulation process, i.e. formation of a fibrin clot.
Disadvantages of the apparatus include: inconvenience of the used dish for high efficiency studies; use of unstable illumination with a mercury lamp, which doesn't allow taking precise measurements; formation of gas bubbles in the area where the process is recorded while the test samples are heated, distorting the fluorescence signal.
The method does not allow modeling in-vitro systems that are similar in their physiological properties to in-vivo systems, and does not allow a more accurate diagnosis of disorders in the blood coagulation system.
Moreover, the above method doesn't allow adequate investigation of the spatial kinetics of coagulation factors, first of all thrombin, during the process of fibrin clot growth, and does not provide the opportunity to evaluate the role of coagulation factor in the different phases of the blood coagulation process in a heterogeneous system.
Thus there is a clear need to improve existing methods and devices for better determination of blood and blood components' coagulation properties.